Method for Calibrating a Transfer Function of a Magnetic Sensor

ABSTRACT

A method for calibrating a transfer function of a magnetic sensor (MS) on a substrate (SBSTR) in which sensor (MS) the presence of magnetizable objects (SPB) can be detected by magnetizing the objects (SPB) by a magnetic field (H) delivered by a magnetic field generator (WR 1 , WR 2 ) and in which the transfer function is defined as the transfer from an electrical input signal (I in ) for generating the magnet field H, via magnetic stray field (SF) radiated by the objects (SPB) when magnetized, to an electrical output signal (I out ) delivered by the sensor (MS), comprising the steps of: putting sample fluid on the substrate (SBSTR), the sample fluid comprising a large amount of the magnetizable objects (SPB), attracting part of the magnetizable objects (SPB) towards the magnetic sensor (MS), activating the electrical input signal (I in ), thereby generating the magnet field (H), measuring the electrical output signal (I out ) as a response to the electrical input signal (I in ), calculating the transfer function from the electrical input and output signals (I in , I out ).

The invention relates to a method for calibrating a transfer function of a magnetic sensor in which sensor the presence of magnetizable objects can be detected by magnetizing the objects by a magnetic field delivered by a magnetic field generator and in which the transfer function is defined as the transfer from an electrical input signal for generating the magnet field, via magnetic stray field radiated by the objects when magnetized, to an electrical output signal delivered by the sensor.

The invention further relates to a magnetic sensor which performs said calibrating and to the use of such a sensor in a biochip for e.g. molecular diagnostics, biological sample analysis or chemical sample analysis.

The introduction of micro-arrays or biochips is revolutionizing the analysis of samples for DNA (desoxyribonucleic acid), RNA (ribonucleic acid), nucleic acids, proteins, cells and cell fragments, tissue elements, etcetera. Applications are e.g. human genotyping (e.g. in hospitals or by individual doctors or nurses), medical screening, biological and pharmacological research, detection of drugs in saliva. The aim of a biochip is to detect and quantify the presence of a biological molecule in a sample, usually a solution.

Biochips, also called biosensors, biosensor chips, biological microchips, gene-chips or DNA chips, consist in their simplest form of a substrate on which a large number of different probe molecules are attached, on well defined regions on the biochip, to which molecules or molecule fragments that are to be analysed can bind if they are matched. For example, a fragment of a DNA molecule binds to one unique complementary DNA (c-DNA) molecular fragment. The occurrence of a binding reaction can be detected, e.g. by using fluorescent markers that are coupled to the molecules to be analysed. As an alternative to fluorescent markers magnetizable objects can be used as magnetic markers that are coupled to the molecules to be analysed. It is the latter type of markers which the present invention is dealing with. In a biochip said magnetizable objects are usually implemented by so called superparamagnetic beads. This provides the ability to analyse small amounts of a large number of different molecules or molecular fragments in parallel, in a short time. One biochip can hold assays for 10-1000 or more different molecular fragments. It is expected that the usefulness of information that can become available from the use of biochips will increase rapidly during the coming decade, as a result of projects such as the Human Genome Project, and follow-up studies on the functions of genes and proteins. A general explanation of the functioning of the biochip has already been described in the international patent application of the present applicant published as WO 03/054523 A2.

A biochip consisting of an array of sensors (e.g. 100) based on the detection of superparamagnetic beads may be used to simultaneously measure the concentration of a large number of different molecules (e.g. protein, DNA, drugs of abuse, hormones) in a sample fluid (e.g. a solution like blood or saliva). The sample fluid comprises a target molecule species or an antigen. Any biological molecule that can have a magnetic label (marker) can be of potential use. The measurement may be achieved by attaching a superparamagnetic bead to the target, magnetizing this bead with an applied magnetic field, and using (for instance) a Giant Magneto Resistance (GMR) sensor to detect the stray field of the magnetized beads.

In the current patent application focus is on a biochip based on (integrated) excitation of superparamagnetic nanoparticles. However also the application in other magneto resistive sensors like Anisotropic Magneto Resistor (AMR) and Tunnel Magneto Resistor (TMR) is part of the invention. The magnetic field generator may comprise a current flowing in a wire which generates a magnetic field, thereby magnetizing a superparamagnetic bead. The stray field from the superparamagnetic bead introduces an in-plane magnetization component in the GMR, which results in a resistance change.

In order to get an unambiguous relation between the number of immobilized beads and the detected signal from the GMR sensors, the total transfer function of the biochip should be calibrated. Said transfer function depends on the device geometry, the properties of the superparamagnetic beads, the properties of the GMR sensor and the signal processing electronics (e.g. pre-amplifier).

Parameters like

Geometry tolerances

Magnetic properties of the superparamagnetic beads

GMR stack tolerances, e.g. thickness, magnetic properties

External magnetic fields

IC (Integrated Circuit) tolerances in signal processing circuitry

Aging effects

Temperature

may have a strong influence on the transfer function. Furthermore the common practice of applying a calibration fluid (usually a liquid) for calibration will complicate the design of the biochip, especially the design of the micro fluids part. The calibration fluid is for instance artificial blood which should be kept in a refrigerator or a freezer until it is needed for calibrating one of the sensors on the biochip which can then serve as a reference sensor for the other sensors during the analysis of for instance (real) blood samples. Furthermore the calibration fluid may be stored in a dedicated container integrated in the biosensor cartridge. Prior to the actual assay the calibration fluid is guided over the sensor surface. This requires a complicated cartridge design. Thus the use of a calibration fluid prior to the actual measurement is both complicate and expensive. There is thus a general need to develop methods for analyzing the sample fluids without the need of an additional fluid which function as a calibration fluid.

It is therefore an object of the invention to provide a method for calibrating the magnetic sensor without the need of a calibration fluid. In order to achieve this object the invention provides a method for calibrating a transfer function of a magnetic sensor on a substrate in which sensor the presence of magnetizable objects can be detected by magnetizing the objects by a magnetic field delivered by a magnetic field generator and in which the transfer function is defined as the transfer from an electrical input signal for generating the magnet field, via magnetic stray field radiated by the objects when magnetized, to an electrical output signal delivered by the sensor, comprising the steps of:

-   putting sample fluid on the substrate, the sample fluid comprising     magnetizable objects, -   attracting (at least) part of the magnetizable objects towards the     sensor, -   activating the electrical input signal, thereby generating the     magnet field, -   measuring the electrical output signal as a response to the     electrical input signal, and -   calculating the transfer function from the electrical input and     output signals.

The sample fluid is for instance a blood or a saliva sample, comprising a target particle (molecule), taken from a human or animal body which is supplemented by magnetizable objects, e.g. superparamagnetic beads, which bind to the target particles. Usually there is a plurality of sensors integrated on the same substrate of a single biochip. Although possible, it is not necessary to calibrate all these sensors by the abovementioned method. Basically it is only necessary to calibrate a single (reference) magnetic sensor. The thus acquired transfer function of this reference sensor can then also be used as the transfer function for the other sensors. This is because of the fact that all sensors are fabricated on the same substrate, and thus electronic parameters of the sensors match with each other. Alternatively it is also possible to determine the transfer functions of a multiple of sensors and use the outcome to determine an average transfer function of the multiple of sensors, which average transfer function can then also serve as the transfer function for the remaining sensors. Hereby the overall accuracy (average accuracy of all sensors on the same substrate) of the calibration is increased. Of course in this situation the part (amount) of the attracted magnetizable objects should be large enough to at least partly cover the active areas (probe areas) of the multiple of magnetic sensors. Said transfer function is used as a reference for the bio-measurements to be performed after the calibration. It is also possible to only use a single sensor which is first calibrated by the inventive method, thus with the use of the sample fluid, and after calibration the bound target molecules are released (e.g. by (magnetically) washing it away), and then putting a sample fluid (which can be from the same sample fluid batch, thus no additional human intervention is needed) on the single sensor.

Thus the transfer function is calibrated prior, or during the actual bio-measurement without the use of a calibration fluid. The transfer function may be calibrated for a large frequency range in order to characterize the complete biochip system like capacitive crosstalk, bead properties (magnetic susceptibility χ, relaxation time τ_(nee1), rotational parameters), pre-amplifier gain etceteras.

In an embodiment the beads are attracted in a well-defined way to a sensor surface. For this purpose the surface of at least one reference-sensor (comprising GMR plus field generating wires) in an array of sensors on a biochip is not (in contrast as indicated in FIGS. 1 and 4) covered by anti-bodies, hence no target molecules will immobilize to said reference-sensor. After applying magnetic beads to the sample fluid, said beads are attracted in a well-defined way towards the reference-sensor by field generating wires near the reference sensor. The GMR response from the reference sensor is indicative to the transfer function of the sensor system. If the reference-sensor surface is sufficiently covered by beads, then the reference sensor is shielded from free moving beads, that is to say the not attracted beads, near the sensor, which may influence the measurement of the transfer function. Alternatively, free moving beads above the reference-sensor may (magnetically) be washed away. In this embodiment it is assumed that the GMR stack, the geometry and the magnetic beads are equal for every sensor on the biochip. An advantage of this method is the fact that beads from the same ‘micro-batch’ are used for calibration as for the actual bio-measurement.

In another embodiment a high affinity binding takes place between beads in the sample volume and at least one sensor surface. In this way a high areal bead density, which saturates the sensor, is realized at a sensor surface. As an example steptavidine coated beads and biotine on the sensor surface.

In another embodiment, superparamagnetic beads are applied to the reference-sensor during production. This can be achieved by either e.g. spotting (like ink-jet spotting) a well defined surface density concentration of beads or a well defined volume density of beads.

These beads may be utilized for calibration of the transfer function. If the sensor is shielded for free moving beads in the sample fluid, which is the case if the bead coverage is large enough, the transfer function may also be stabilized during the actual bio-measurement.

The invention will be further elucidated with reference to the accompanying drawings, in which:

FIG. 1 shows a biochip comprising a substrate and a plurality of magnetic sensors,

FIG. 2 shows an embodiment of a magnetic sensor with integrated magnetic field excitation;

FIG. 3 shows the resistance of the GMR as a function of the magnetic field component in the direction in which the layer of the GMR is sensitive to magnetic fields; and

FIG. 4 shows a method for calibrating the transfer function of the magnetic sensor as shown in FIG. 2.

The drawings are only schematic and non-limiting. In the drawings the size of some of the elements may be exaggerated and not drawn on scale and serve only for illustrative purposes. The description to the Figures only serve to explain the principles of the invention and may not be construed as limiting the invention to this description and/or the Figures.

FIG. 2 shows an embodiment of a magnetic sensor MS on a substrate SBSTR. A single or a multiple of such (a) sensor(s) may be integrated on the same substrate SBSTR to form a biochip BCP as is schematically indicated in FIG. 1. The magnetic sensor MS comprises a magnetic field generator which is preferably integrated in the substrate SBSTR e.g. by a first current conducting wire WR₁. It may also comprise a second (or even more) current conducting wire WR₂. Also other means in stead of a current conducting wire may be applied to generate the magnetic field H. The magnetic field generator may also be located outside (external excitation) the substrate SBSTR. A magnetoresistive element, for example a giant magnetoresistive resistor GMR, is integrated in the substrate SBSTR to read out the information gathered by the biochip BCP, thus to read out the presence or absence of target particles TR via magnetizable objects thereby determining or estimating an areal density of the target particles TR. The magnetizable objects are preferably implemented by so called superparamagnetic beads SPB. Binding sites BS which are able to selectively bind a target TR are attached on a probe element PE. The probe element PE is attached on top of the substrate SBSTR.

The functioning of the magnetic sensor MS or more generally of the biochip BCP is as follows. Each probe element PE is provided with binding sites BS of a certain type. Target sample TR is presented to or passed over the probe element PE, and if the binding sites BS and the target sample TR match, they bind to each other. The superparamagnetic beads SPB are directly or indirectly coupled to the target sample TR. The superparamagnetic beads SPB allow to read out the information gathered by the biochip BCP. Superparamagnetic particles are suspended in a (polymer) binder or matrix of which at zero applied magnetic field the time-averaged magnetization is zero due to thermally induced magnetic moment reversals that are frequent on the time scale of the magnetization measurement. The average reversal frequency is given by $v = {v_{0}\exp\frac{- {KV}}{kT}}$ where KV (with K the magnetic anisotropy energy density and V the particle volume) is the energy barrier that has to be overcome, and v₀ is the reversal attempt frequency (typical value: 10⁹ s⁻¹).

The magnetic field H magnetizes the superparamagnetic beads SPB which as a response generate a stray field SF which can be detected by the GMR. Although not necessary the GMR should preferably be positioned in a way that the parts of the magnetic field H which passes through the GMR is perpendicular to the sensitive direction of the layer of the GMR. A total external (that is to say not internal in the GMR) field for which the GMR is sensitive is indicated by H_(ext) in FIG. 2.

With the assumption that the biochip BCP is horizontally positioned (thus perpendicular to the force of gravity) the GMR is positioned in a manner that it is only or mainly sensitive to magnetic fields having a horizontal component. Therefore the GMR is not sensitive to the magnetic field H because if it passes perpendicular to the GMR and has thus no horizontal component. In contrast the stray field SF does have a horizontal component and will thus generate a difference in the resistance value of the GMR. By this an electrical output signal (e.g. generated by a current change through the GMR when biased by a DC voltage, not shown in FIG. 1) can be delivered by the sensor MS which is a measure for the amount of targets TR.

FIG. 3 shows the resistance of the GMR as a function of the magnetic field component H_(ext). It is to be noted that the GMR sensitivity $s_{GMR} = \frac{\mathbb{d}R_{GMR}}{\mathbb{d}H_{ext}}$ is not constant but depends on H_(ext).

In the sensor MS as shown in FIG. 2, in stead of the giant magnetoresistive GMR any other means which have a property (parameter) which depends on magnetic field such as certain types of resistors like a tunnel magnetoresistive (TMR) or an anisotropic magnetoresistive (AMR) can be applied. In an AMR, GMR or TMR material, the electrical resistance changes when the magnetization direction of one or more layers changes as a result of the application of a magnetic field. GMR is the magnetoresistance for layered structures with conductor interlayers in between so-called switching magnetic layers, and TMR is the magneto-resistance for layered structures comprising magnetic metallic electrode layers and a dielectric interlayer.

In GMR technology, structures have been developed in which two very thin magnetic films are brought very close together. A first magnetic film is pinned, which means that its magnetic orientation is fixed, usually by holding it in close proximity to an exchange bias layer, a layer of antiferromagnetic material that fixes the first magnetic film's magnetic orientation. A second magnetic layer or free layer, has a free, variable magnetic orientation. Changes in the magnetic field, in the present case originating from changes in the magnetization of the superparamagnetic particles SPB, cause a rotation of the free magnetic layer's magnetic orientation, which in turn, increases or decreases the resistance of the GMR structure. Low resistance generally occurs when the sensor and pinned layers are magnetically oriented in the same direction. Higher resistance occurs when the magnetic orientations of the sensor and pinned layers (films) oppose each other.

TMR can be observed in systems made of two ferromagnetic electrode layers separated by an isolating (tunnel) barrier. This barrier must be very thin, i.e., of the order of 1 nm. Only then, the electrons can tunnel through this barrier. This is a quantum-mechanical transport process. The magnetic alignment of one layer can be changed without affecting the other by making use of an exchange bias layer. Changes in the magnetic field, in the present case originating from changes in the magnetization of the superparamagnetic particles SPB, cause a rotation of the sensor film's magnetic orientation, which in turn, increases or decreases resistance of the TMR structure.

The AMR of ferromagnetic materials is the dependence of the resistance on the angle the current makes with the magnetization direction. This phenomenon is due to an asymmetry in the electron scattering cross section of ferromagnet materials.

FIG. 4 shows a method for calibrating the transfer function of the magnetic sensor (MS). Sample fluid is put on the substrate SBSTR. The sample fluid comprises superparamagnetic beads SPB. It is a major advantage of the application of the current inventive method that calibration can be applied with only using sample fluid, thus no need for the use of for instance artificial blood as a calibration fluid. Thus the sample fluid from the same batch can be used for both the calibration and the biomeasurements after the calibration.

Accurate calibration can be acquired for instance by putting a large amount of the sample fluid to completely cover the complete active area of the magnetic sensor MS with a very thin layer of superparamagnetic beads SPB. The superparamagnetic beads SPB are magnetically attracted to the active area (probe elements PE) of the sensor MS by a magnetic force caused by for instance the magnetic fields generated by the first and second wires WR₁, WR₂. Alternatively it is also possible to generate this force by a further magnetic field (not shown in FIG. 4). It is to be noted that the exact amount of superparamagnetic beads SPB need not be known. This is because the amount of detected stray field SF sensed by the sensor GMR is influenced approximately only by the superparamagnetic beads SPB close to the surface. This is because the distance from higher positioned beads SPB is larger and thus the influence is smaller. This effect is further enhanced by the fact that the magnetic stray field SF of the higher positioned superparamagnetic beads SPB is magnetically shielded by the lower (thus closest to the substrate SBSTR) positioned superparamagnetic beads SPB.

Another method for acquiring accurate calibration is controlling the amount of the superparamagnetic beads SPB. Under the assumption that the bead volume density is known, the controlling can be done by performing the magnetically attraction of the beads for a fixed (and known) amount of time. Alternatively a controlled amount of superparamagnetic beads SPB can be reached by sedimentation. In the latter case a fixed (and known) amount of time should be awaited in which sedimentation occurs. Also in the latter case it is assumed that the bead volume density is known.

The sensor MS comprises an electronic circuit CRT which is coupled to the GMR to measure its resistance and more specifically its changes in resistance. By way of example this circuit CRT also provides an electrical input signal, which may be a current or a voltage, through or at the first electrical wire WR₁ (and possibly also through or at the second wire WR₂). In this example the electrical input signal is represented by an input current I_(in). The input current I_(in) may also be supplied by other means for instance means which are positioned outside the biochip CHP. The input current I_(in) generates a magnetic field H (not indicated in FIG. 4, but see FIG. 2). The superparamagnetic beads SPB of said very thin layer (at the lower side) generates a stray field (not indicated in FIG. 4) similar to the stray field SF as indicated in FIG. 2. Thus a horizontal component of this stray field (see H_(ext) in FIG. 2) causes a change in resistance of the GMR. This change is measured by the circuit CRT. As a response to this the circuit CRT supplies a signal to an amplifier G which responses by delivering an electrical output signal in the form of an output signal I_(out). The value of the output current I_(out) divided by the value of the input current I_(in) may be used as the reference transfer function of the sensor. However the circuit CRT may also be used to tune this transfer function to be equal to one (for a certain frequency) by supplying a certain gain adaptation value GAV to the amplifier G so that the gain of the amplifier G is lowered or increased by the appropriate amount. This may also be carried out for more than one frequency, e.g. a range of frequencies, of the input signals I_(in). The calibration is now completed.

After completion of the calibration a small amount of sample fluid, e.g. a drop of blood, in which superparamagnetic beads SPB are attached to the target molecules TR, is put on a magnetic sensor MS, the input current I_(in) is activated, an output current I_(out) is generated and measured by a measuring system MRS thereby measuring the amount of immobilized superparamagnetic beads SPB and thus the amount of target molecules TR. The output current I_(out) can be supplied to the measuring system MRS via a bonding pad PB. Basically numerous sensors (not shown in the Figures) on the same substrate SBSTR (so in the same biochip BCP) can be used all having “loaded” the gain adaptation value GAV to its amplifier G. Thus basically the sensor MS which was previously calibrated serve as a reference sensor for all the other sensors MS in the same biochip BCP.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and those skilled in the art will be capable of designing alternative embodiments without departing from the scope of the invention as defined by the appended claims. In the claims, any reference signs placed in parentheses shall not be construed as limiting the claims. The words “comprising” and “comprises”, and the like, do not exclude the presence of elements other than those listed in any claim or in the application as a whole. The singular reference of an element does not exclude the plural reference of such elements. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures can not be used. 

1. A method for calibrating a transfer function of a magnetic sensor (MS) on a substrate (SBSTR) in which sensor (MS) the presence of magnetizable objects (SPB) can be detected by magnetizing the objects (SPB) by a magnetic field (H) delivered by a magnetic field generator (WR₁, WR₂) and in which the transfer function is defined as the transfer from an electrical input signal (I_(in)) for generating the magnet field H, via magnetic stray field (SF) radiated by the objects (SPB) when magnetized, to an electrical output signal (I_(out)) delivered by the sensor (MS), comprising the steps of: putting sample fluid on the substrate (SBSTR), the sample fluid comprising magnetizable objects (SPB), attracting part of the magnetizable objects (SPB) towards the sensor (MS), activating the electrical input signal (I_(in)), thereby generating the magnet field (H), measuring the electrical output signal (I_(out)) as a response to the electrical input signal (I_(in)), and calculating the transfer function from the electrical input and output signals (I_(in), I_(out)).
 2. A method as claimed in claim 1, characterized in attracting the part of magnetizable objects (SPB) by a force acting on the part of magnetizable objects (SPB) which is caused by a gradient in the magnetic field (H).
 3. A method as claimed in claim 1, characterized in applying a further magnetic field causing a force acting on the part of magnetizable objects (SPB) caused by a gradient in the further magnetic field.
 4. A method as claimed in claim 3, characterized in that a current is flowing through a wire in the neighborhood of, or integrated in, the sensor (MS), thereby generating the further magnetic field.
 5. A method as claimed in claim 1, characterized in having an additional step after the step of attracting part of the magnetizable objects towards the sensor (MS), the additional step being: washing away a free moving part of the objects (SPB).
 6. A method as claimed in claim 5, characterized in that the washing away of the free moving part of the objects (SPB) is being carried out by magnetically washing away the free moving part of the objects (SPB).
 7. A method for calibrating a transfer function of a magnetic sensor (MS) on a substrate (SBSTR) in which sensor (MS) the presence of magnetizable objects (SPB) can be detected by magnetizing the objects (SPB) by a magnetic field (H) delivered by a magnetic field generator (WR₁, WR₂) and in which the transfer function is defined as the transfer from an electrical input signal (I_(in)) for generating the magnet field, via magnetic stray field (SF) radiated by the objects (SPB) when magnetized, to an electrical output signal (I_(out)) delivered by the sensor (MS), comprising the steps of: shooting part of the magnetizable objects (SPB) towards the sensor (MS), activating the electrical input signal (I_(in)), thereby generating the magnet field (H), measuring the electrical output signal (I_(out)) as a response to the electrical input signal (I_(in)), and calculating the transfer function from the electrical input and output signals (I_(in), I_(out)).
 8. A method for calibrating a transfer function of a reference magnetic sensor (MS) on a substrate (SBSTR) whereby during the fabrication process of the reference sensor (MS) magnetizable objects (SPB) are attached and in which reference sensor (MS) the presence of the magnetizable objects (SPB) can be detected by magnetizing the objects (SPB) by a magnetic field (H) delivered by a magnetic field generator (WR₁, WR₂) and in which the transfer function is defined as the transfer from an electrical input signal (I_(in)) for generating the magnet field (H), via magnetic stray field (SF) radiated by the objects (SPB) when magnetized, to an electrical output signal (I_(out)) delivered by the reference sensor (MS), comprising the steps of: activating the electrical input signal (I_(in)), thereby generating the magnet field (H), measuring the electrical output signal (I_(out)) as a response to the electrical input signal (I_(in)), and calculating the transfer function from the electrical input and output signals (I_(in), I_(out)).
 9. A magnetic sensor (MS) on a substrate (SBSTR) in which the presence of magnetizable objects (SPB) can be detected, and presented by an electrical output signal (I_(out)), by magnetizing the objects (SPB) by a magnetic field (H) delivered by a magnetic field generator (WR₁; WR₂), comprising calibration means for calibrating a transfer function which is defined as the transfer from an electrical input signal (I_(in)) for generating the magnet field (H), via magnetic stray field (SF) radiated by the objects (SPB) when magnetized, to the electrical output signal (I_(out)), for executing the method steps as defined in claim
 1. 10. A biochip (BCP) comprising a plurality of magnetic sensors (MS) wherein at least one of the sensors is a sensor as defined in claim
 9. 